Don't forget to do seeding with the native crystals: it's often more about nucleation than growth. Phx
Sent from tiny silly touch screen ----- Reply message ----- From: "Frederic VELLIEUX" <frederic.velli...@orange.fr> Date: Mon, Jun 13, 2011 07:37 Subject: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> I've had exactly the same with an enzyme from Trypanosoma brucei, native enzyme gives ca. 2 A data, a search around the crystallization conditions for the Se-Met enzyme returns absolutely nothing. What I should do is to search for new crystallisation conditions (which I won't do: trypanosome means no-one will provide any funding for the project nowadays). So if you do not get any crystals from your derivatized protein starting from the crystallisation conditions of your native protein, then you must start a new crystallisation screening process right from the start. Remember that introducing Se-Mets can change the surface of your protein; it can also introduce an equilibrium between several forms of the protein, whereas you only had one form for the native enzyme - so you may need to try to purify (rapidly) further. If this de novo crystallisation fails, then you need to try alternative phasing methods (such as heavy atom soaks or co-crystallisation with heavy atoms). Fred. > Message du 13/06/11 07:53 > De : "atul kumar" > A : CCP4BB@JISCMAIL.AC.UK > Copie à : > Objet : [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation > > ---------- Forwarded message ---------- > From: Dilip Kumar > Date: Mon, Jun 13, 2011 at 11:21 AM > Subject: Fwd: Regarding Sel-Met containing proteing crystallisation > To: atulsingh21...@gmail.com > > > > > ---------- Forwarded message ---------- > From: Dilip Kumar > Date: Sat, Jun 11, 2011 at 6:09 PM > Subject: Regarding Sel-Met containing proteing crystallisation > To: CCP4BB@jiscmail.ac.uk > > > Dear all > > I have got protein crystals,crystallisation condition (LiCl, PEG and > HEPES) .Crystals of native protein have been successesfully reproduced but > when i tried to reproduce these crystals with protein having Met replaced by > Sel-Met, i could not get any crystal.I tried crystallisation trials by > varying pH and PEG concentration and diffferent drop ratio but i could not > get any hit.Please suggest me what could be the possible reasons behind it? > And also suggest the other variables that i can try ? > thanks > With Regards > > -- > Dilip Tiwari > Graduate Student > Structural Biology Unit > Institute of Genomics & Integrative Biology > Delhi-07 > > > > -- > Dilip Tiwari > Graduate Student > Structural Biology Unit > Institute of Genomics & Integrative Biology > Delhi-07 >
